| [Background and Objective]The two difficult problems of severe burns are Lack of skin donor sites and delayed time of skin wound healing,delayed time of skin wound healing can result in a consuming state of body in a long period.This can lead to the descent of resistance abilities of body,easy getting infections and multiple organ failure.Since the invention of microskin transplantation technique,utilizing a small quantity of splitthickness skin graft to make into miroskin tissue particles,the extinction of wound surface be become into reality.This bring a revolutionary breakthrough in repairing severe burns wound surface. There are some shortcomings of microskin transplantation in this technique:1.The skin by microskin extended is lack of integral dermis, very thin and easy to ulcerate,be prone to become wound surface again; 2.After the wound surface healing of applying microskin tissue particles transplantation,a mass of hyperplasia scar come into being, affected the appearance and function of the body.3.The surviving of microskin on wound surface is not insure some time,and will not get a good result.The coming forth of stem cell technique bring a new way in repairing the wound surface.The stem cell can be provided with strong hyperplastic ability.In a certain condition,it can be differentiated into kinds of functional cells.Base on the phase of cell development,stem cell be divided into two categories:embryonic stem cell and adult stem cell. The research in embryonic stem cells is restricted by its limited source and ethical controversy,so research on adult stem cells attracts more attentions.Bone marrow mesenchymal stem cell is a kind of adult stem cell,derived from mesoderm.The mechanism of BMSCs repairing wound surface lie in the following ways:1.Stem cells have self-regenerate ability and strong differentiate ability.Adapt to the microenvironment change on wound,it can be differentiated into kinds of cells and integrated into local tissues according to the needs of the body.2.The enabled BMSCs in wound surface can act as a micro-bioreactor,it can secrete kinds of growth factor and cell factor,accommodate the proliferation and differentiation of local tissue,the formation of cell matrix,the formation of vascular et al;3.Once the body has been damaged, at the same time of starting process of inflammation reaction,BMSCs can migrate into damaged tissue and join the repairing of the tissue.This project bases on the principle of the proliferation and differentiation of BMSCs.Transplanted the BMSCs and microskin tissue into fresh wound surface,utilize cell culture immunological marking and reaction,molecular biology histopathology microcosmic detection, monitoring the proliferation and differentiation of BMSCs and the hyperplasia and extending of microskin particles,The experimental data will provide the evidences for the following way:1.How are the BMSCs acted as micro-bioreactor in fresh wound surface, and how are the orientation of proliferation and differentiation and the way of forming cell matrix.2.Whether BMSCs can attach to and invade into and migrate into microskin tissue particles,proliferating and differentiating,and improve the surviving of microskin tissue particles in fresh wound surface.3.Whether the BMSCs and microskin tissue particles can accelerate the healing of wound surface,and improve the healing quality of wound surface.[Methods]Part one Isolation and identification of BMSCs of male F344 rat in vitro1.Isolation,culture and passage:break off the neck and execute male F344 rat,douche the bone marrow from femur,tibia and humerus, inoculate these cells into culture flask,replace the culture media after 48 hours,remove the non-adherent cells.Culture media was changed every 48 hours.Cells were passaged with ratio of 1:2 when almost confluent.Passage 3 cells were used for further experiment.2.Cell growth kinetics:cell proliferation activity of passage 3 and passage 5 cells were measured,cell proliferation curve were be drawn.3.Cell cycle examination of BMSCs:active growing passage 3 cells were detached by 0.25%trypsin and made into single cell suspension,stained for propidium iodide,then passed through flow cytometer.4.Cell surface marker examination of BMSCs by flow cytometer:active growing passage 3 cells were detached by 0.25%trypsin and made into single cell suspension,take 1ml cells which concentration is 1×10~6/ml and stain for CD29,CD34,CD44 and CD90 labelled with FITC,then measured by flow cytometer.5.Lipogenesis and osteogenesis induction and characterization: passaged 3 cells were induced with lipogenetic reagent and osteogenetic reagent,stained for Oil Red 0 and Alizarin Red S, measured by microscope.Part two experimental study on transplantation of bone mesenchymal stem cell and microskin tissue into wound surface1.take passage 5 cells labeled with 5-Brdu(10μmol/L) for 48 hours.2.Take full thickness skin graft of Wistar rat,cut into size 4×4cm~23.48 F344 female rat take into four groups:group A transplant bone mesenchymal stem cell and microskin tissue particles into wound surface;group B transplant microskin tissue particles into wound surface;group C transplant bone mesenchymal stem cell into wound surface;group D injected with physiological saline solution.Forming a wound surface of size 4×4cm~2,take a quarter of full thickness skin graft cut into microskin tissue particles,lay on surface of xenoma skin,suture and fix on the surface.4.Opened up the xenoma skin 14 days post-operation,observed and record healing rate of wound surface;Observed and recorded healing time of wound surface;Record the shrinkage rate of wound surface.5.On the day of wound surface healing,execute the rat,took the skin specimen,preserved the specimen on refrigerator of -80℃.Part three experimental study on proliferation and differentiation of the bone mesenchymal stem cell on wound surface 1.hybridization in situRat sry gene was labelled in freezing section,measured in optical microscope.2.Examination of immune fluorescence histochemistryFreezing sections were stained for Cy3 conjugated secondary antibody combined with BrdU antibody,FITC conjugated secondary antibody combined with FⅧ,Keratin,actin,nestin and S-100 antibody,analysed with fluorescence microscope.[Result]Part one Isolation and identification of BMSCs of male F344 rat in vitro1.After the full bone marrow cell suspension was inoculated in a culture flask,a small amount of bone marrow mononuclear cells adhere to the flask 24 hours after planting.After 48 hours,suspending cells were discarded.In 96 hours,suspending cells were decreased obviously,the cells were be growing with clone formation,reaching homogeneous confluence in 12 hours.In 10 days,the cells were fusion,cannot see the suspending cells basically.2.Flow cytometer analysis showed that about 20.19%of cells are in S+G2+M phase,79.81%are in Go/G phase,and only small numbers are in active proliferation state.3.Identification of BMSCs superficial marker:79.15%cells were CD29 positive cells;9.10%cells were CD34 positive cells;89.47%cells were CD44 positive cells;72.34%cells were CD90 positive cells.4.lipogenesis and osteogenesis induction and characterization:After 14 days lipogenesis induction,and stained for Oil Red O,many tiny lipid droplet can be seen in cytoplasm,In 21 days,lipid droplet in cytoplasm fused,like vacuole,osteogenesis induction stained for Alizarin Red S,in 14 days,calcium salt deposition can be seen;In 21 days,brown red mass can be seen among the induction cells.Part two experimental study on transplantation of bone mesenchymal stem cell and microskin tissue particles into wound surface1.Healing rate of wound surface:xenoma skin was opened in 14 days,wound surface of group A had been healed basically,healing rate of wound surface was 85.77±3.47%;healing rate of wound surface was 62.16±4.39%;The wound surface of group C,group D was flesh bud tissue healing rate of wound surface was 30.44±1.73%,29.96±1.33% respectively;The healing of wound surface was own to shrinkage of wound surface.Comparison between group A and group B existed in Significant difference(P＜0.001);Welch=1028.700,P＜0.001;Comparison between group C and group D has no difference(P=0.898)2.Healing time of wound surface:The healing time of wound surface of four groups is 17.17±1.64,20.33±2.38,23.08±1.31,25.16±1.53 respectively,Comparison between group A and group B existed in Significant difference(P＜0.001),Comparison between group C and group D existed in Significant difference(P＜0.01).3.shrinkage rate of wound surface:The shrinkage rate of wound surface of four groups is 36.89±1.94%,39.56±2.93%,92.51±1.44%,92.40±1.91%respectively,Comparison between group A and group B existed in Significant difference(P＜0.01);Comparison between group C and group D has no difference(P=0.903)Part three experimental study on proliferation and differentiation of the bone mesenchymal stem cell on wound surface 1.Examination in situ hybridization:In the microskin specimen sections of group A,many cells which nucleolus is brown can be seen among hair follicle.A few brown nucleolus cells were scattering in the dermis;A few brown nucleolus cells were scattering in the healing scar tissue specimen sections of group C;Few these cells could be seen among specimen sections of group B and group D.2.Examination of immune fluorescence histochemistry:1) Red fluorescence of labeled BrdU and green fluorescence of labelled Keratin can be seen among hair follicle and epidermis base layer in group A specimen sections,and existed in the seem position;Green fluorescence of labelled Keratin can be seen among hair follicle and epidermis base layer in group C specimen sections,but no red fluorescence of labelled BrdU can be seen in group C specimen sections; In group D specimen sections red fluorescence of labeled BrdU and green fluorescence of labelled Keratin can not be seen.2) Red fluorescence of labelled BrdU and green fluorescence of labelled FⅧcan be seen in the dermis of group A and healed scar of group C specimen sections,and formed a structure like vascular,and existed in the seem position;In the dermis of group B and healed scar group D specimen sections green fluorescence of labelled FⅧcan be seen, red fluorescence of labelled BrdU cannot be seen.3) Red fluorescence of labelled BrdU and green fluorescence of labelled Actin can be seen in the dermis of group A and healed scar group C specimen sections,and existed in the seem position;In the dermis of group B and healed scar of group D specimen sections green fluorescence of labelled Actin can be seen,red fluorescence of labelled BrdU cannot be seen.4) Red fluorescence of labelled BrdU and green fluorescence of labelled Nestin can be seen scattering in the dermis of group A and healed scar group C specimen sections,and existed in the seem position; Green fluorescence of labelled Nestin can be seen in the dermis in group C specimen sections,but no red fluorescence of labelled BrdU can be seen in group C specimen sections;In group D specimen sections red fluorescence of labeled BrdU and green fluorescence of labelled Nestin can not be seen.5) Red fluorescence of labeled BrdU and green fluorescence of labelled S-100 can be seen scattering in the dermis of group A and healed scar group C specimen sections,and existed in the seem position;Green fluorescence of labelled Nestin can be seen in the dermis in group C specimen sections,but no red fluorescence of labelled BrdU can be seen in group C specimen sections;In group D specimen sections red fluorescence of labeled BrdU and green fluorescence of labelled Nestin can not be seen.[Conclusion]1.Isolated BMSCs from rat bone marrow successfully.2.The BMSCs we isolated have multi-potential differentiation ability, accord with characteristic of adult stem cell.3.The BMSCs we transplanted into wound surface promoted the survival of microskin tissue particles and accelerated the healing of wound surface.4.The BMSCs we transplanted into wound surface affected by the microenvironment coming from wound surface and microskin tissue, maybe differentiated into vascular endothelial cells,epidermal cell, myofibroblast,peripheral nerve tissue cell.
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